A plasma membrane glycoprotein, found in the extracellular matrix of normal eukaryotes but absent following transformation, is also present in substantial quantities in normal plasma. The protein has been designated fibronectin and has been shown to mediate the adhesion of cells to a collagen substratum in vitro. As a means of determining an in vivo function for this protein we are investigating its chemical structure and adapting the techniques of in vitro cellular adhesion to a more biologically relevant substratum, specifically basement membranes excised directly from experimental animals. These limiting membranes are known to contain substantial levels of fibronectin. It is assumed that antisera directed against fibronectin and known to inhibit cellular adhesion in vitro will provide experimental means of determining the relevance of fibronectin to cellular adhesion in vivo. Subtilisin digestion of fibronectin under specific conditions yields reproducible patterns of digestion products. Following chromatography of the fragments on a gelatin affinity column a major polypeptide of apparent molecular weight 50,000 daltons binds to and is eluted with 4M urea from the column. This fragment is still capable of mediating adhesion of fibroblasts to collagen and therefore contains both the gelatin and cellular binding sites. Further chemical characterization of this fragment has been accomplished.